WO1994020125A1 - Traitement des affections des neurones moteurs a l'aide du facteur de croissance des fibroblastes 5 (fgf-5) - Google Patents

Traitement des affections des neurones moteurs a l'aide du facteur de croissance des fibroblastes 5 (fgf-5) Download PDF

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WO1994020125A1
WO1994020125A1 PCT/EP1994/000764 EP9400764W WO9420125A1 WO 1994020125 A1 WO1994020125 A1 WO 1994020125A1 EP 9400764 W EP9400764 W EP 9400764W WO 9420125 A1 WO9420125 A1 WO 9420125A1
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fgf
survival
motor
motor neurons
motor neuron
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PCT/EP1994/000764
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Richard A. Hughes
Michael Sendtner
Dan Lindholm
Hans F. E. Thoenen
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to AU62841/94A priority Critical patent/AU6284194A/en
Publication of WO1994020125A1 publication Critical patent/WO1994020125A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/50—Fibroblast growth factors [FGF]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method of treating a motor neuron disorder comprising administering to a subject in need of such treatment an effective amount of FGF-5 or a functional derivative of FGF-5 which promotes survival of motor neurons. Also provided are methods of culturing motor neurons, methods of diagnosing motor neuron disorders, assay systems for identifying agents which may be beneficial or toxic to motor neurons, and animal models for motor neuron diseases.
  • MOTOR NEURONS The interaction between motor neurons and muscle cells underlies all voluntary and involuntary body movement by controlling muscle tone and effecting muscular contraction.
  • Motor neurons are traditionally classified as upper motor neurons or lower motor neurons.
  • the cell bodies of upper motor neurons reside in the precentral gyrus of the brain, sending long processes down to form synapses with lower motor neurons in the ventral (anterior) horns of the grey matter of the spinal cord. From the ventral horns of the spinal cord, axon processes of lower motor neurons coalesce to form the ventral roots. These axons eventually terminate on one or more muscle fibers.
  • each lower motor neuron contacts anywhere from a few up to 100- 200 or more muscle fibers to form a "motor unit,” (Adams and Victor, 1985, PRINCIPLES OF NEUROLOGY - McGraw-Hill, Inc., New York, p. 37).
  • Motor neuron disorders result in varying degrees of muscle weakness, causing disabilities ranging from minor deficits in the performance of complex tasks to total paralysis.
  • Lower motor neuron disorders are generally associated with flaccid paralysis and decreased muscle tone.
  • Contiguous groups of muscles, innervated by single nerves or whose motor neurons lie close together in the spinal cord may be affected, and atrophy may be quite profound, including up to about 70-80% of the total muscle bulk.
  • a diseased motor neuron may also become irritable, leading to sporadic discharge of muscle fibers under its control in isolation from other units. This results in a visible twitch, or fasciculation.
  • a wide array of neurological disorders affect motor neurons. Upper motor neurons are predominantly affected by cerebrovascular accidents, neoplasms, infections or traumatic injury. Lower motor neurons, or anterior horn cells (AHC) , while secondarily affected by the above processes, are also subject to a number of disorders primarily featuring AHC loss. Such disorders include amyotrophic lateral sclerosis 5 (ALS) , infantile and juvenile spinal muscular atrophy, poliomyelitis", the post-polio syndrome, hereditary motor and sensory neuropathies and toxic motor neuropathies, such as that caused by vincristine therapy.
  • ALS amyotrophic lateral sclerosis 5
  • poliomyelitis the post-polio syndrome
  • hereditary motor and sensory neuropathies such as that caused by vincristine therapy.
  • ALS also known as Lou Gehrig's disease
  • Lou Gehrig's disease is the most common. (For review, see Williams et al . , 1991, Mayo Clin . Proc . 66:54-82). The initial complaint in most ALS patients is weakness, more commonly of the upper limbs
  • ALS amyotrophic lateral sclerosis
  • Upper motor neurons also become involved and undergo degeneration. While, the brain may appear normal macroscopically, atrophy of the motor and premotor cortices is usually present due to upper 5 motor neuron involvement.
  • Betz cells and other pyramidal cells from the precentral cortex, with consequent reactive gliosis (Hammer et al., 1979, Exp . Neurol . 63:336-346).
  • ALS is more a disease of the elderly (Williams et al . , supra) .
  • the woJJler mouse exhibits forelimb weakness and atrophy in early life due to muscle denervation.
  • Hereditary canine spinal muscular atrophy in the Brittany spaniel is also considered an ALS model (Sack et al . , 1984, Ann . Neurol . 25:369-373; Sillevis et al . , 1989, J . Neurol . Sci . 91:231-258; Bird et al., 1971, Acta Neuropathol . 29:39-50).
  • neuronal survival during this period depends upon access of the developing neurons to specific trophic factors supplied by target tissues.
  • NGF nerve growth factor
  • For example, nerve growth factor (NGF) produced in limiting quantities in the target tissues of peripheral sympathetic and sensory neurons, supports the survival of these cells during critical stages of development.
  • NGF nerve growth factor
  • motor neurons rely upon their target during the embryonic period of naturally- occurring cell death (Hamburger, V., Amer. J. Anat . 202:365-410 (1958); Hollyday et al . , J . Comp. Neurol . 270:311-320 (1976) ) .
  • Extracts of skeletal muscle, the target of motor neurons, promote the survival of motor neurons in culture (Bennet et al . , Brain Res . 290:537-542 (1980); Schnaar et al . , J. Neurosci . 2:204-217 (1981); Calof et al . , Devel . Biol . 106:195-210 (1984); Kaufman et al., J . Neurosci . 5:160-166 (1985); Flanigan et al . , J. Neurochem . 45:1323-1326 (1985); Smith et al . , J. Cell Biol . 202:1608-1621 (1985); Dohrmann et al .
  • CNTF ciliary neurotrophic factor
  • bFGF basic fibroblast growth factor
  • a putative candidate 5 molecule should support motor neuron survival and be appropriately expressed in target tissues.
  • Fibroblast growth factors comprise a family of potent mitogens for both normal diploid fibroblasts and established cell lines (Gospodarowicz, D. et al . , Proc . Nat ' l . Acad . Sci . USA 81:6963 (1984)) .
  • the FGF family comprises acidic FGF, basic 5 FGF, INT-2 (FGF-3) , K-FGF/HST (FGF-4), FGF-5, FGF-6, KGF (FGF-7) , AIGF (FGF-8) and possibly others.
  • FGFs are also mitogenic for a wide variety of normal diploid mesoderm-derived and neural crest-derived cells, including granulosa cells, adrenal cortical 0 cells, chondrocytes, myoblasts, corneal and vascular endothelial cells (bovine or human) , vascular smooth muscle cells, and lens epithelial cells.
  • bFGF Basic FGF
  • bFGF acts as a differentiation factor for various cells, including chondrocytes,- sheep preadipocyte fibroblasts, astrocytes, and oligodendrocytes; bFGF treatment of neural cells induces both neurite outgrowth and ornithine decarboxylase activity. Not all of the effects of bFGF on cell differentiation are stimulatory. For example, bFGF can delay differentiation and fusion of normal diploid myoblasts.
  • FGF also significantly delays the ultimate senescence of various types of cultured cells. When maintained in the absence of bFGF, these cells have a limited life-span, whereas in its presence, they can proliferate for more than 200 generations. In development, bFGF promotes limb regeneration in lower vertebrates, and may play a role in the early development of the nervous system. Basic FGF promotes both the survival and differentiation of nerve cells derived either from the hippocampus or the cortex. FGFs may also have pronounced effects on the proliferation and differentiation of astrocytes and oligodendrocytes during normal development or following a specific pathogenic event.
  • FGF has been used for the treatment of ischemic heart disease (Franco, U.S. Patents 4,296,100 and 4,378,347), increasing blood flow in the heart for sustained periods after myocardial infarction.
  • Nevo et al . U.S. Patent No. 4,642,120
  • Senoo et al . European Patent Publication EP281822
  • a mutein of bFGF which accelerated cell growth in vitro and acted as a healing accelerator for burns and a therapeutic drug for thrombosis.
  • Arakawa 5 et al . European Patent Publication EP320148 disclosed recombinant bFGF analogs with at least one of the biological properties of mammalian bFGF including the induction of neovascularization, re- epithelization and wound repair.
  • bFGF stimulates (a) 0 growth of bone marrow stromal cultures, which are capable of supporting hemopoietic cell growth and differentiation, and (b) myelopoiesis in bone marrow culture (Oliver, L.J. et al . , Growth Factors 3:231-236 (1990); Wilson, E.L. et al . , Blood 77:954-960 (1991)).
  • FGF-3 A protein originally termed FGF-3, but now designated FGF-5, was discovered as the gene product of an oncogene called ORF-2 (Goldfarb, M. et al . , PCT o Publication WO 88/01767, Dec. 1, 1988; see, also, Zhan, X. et al . , Oncogene 1:369-376 (1987); Zhan et al . , Mol . Cell . Biol . 8:3487-3495 (1988); these three references are hereby incorporated by reference in their entirety) .
  • the name "FGF-3" was selected 5 because of the substantial homology to the two already described FGFs (aFGF and bFGF) .
  • the structure of the rearranged transforming FGF- 5 oncogene was similar to other FGF-related genes.
  • the FGF-5 gene was expressed in a human brain stem cDNA library, and several human tumor cell lines contained transcripts which hybridized with an FGF-5 cDNA probe.
  • Transfection of an FGF-5 cDNA clone into NIH-3T3 cells resulted in transformants which expressed the FGF-5 oncogene and secreted a growth factor apparently encoded by this gene.
  • the presumed FGF-5 product also resembled partially purified bFGF in its stimulation of endothelial cell growth.
  • extracts of bacteria expressing the FGF-5 protein contained a heparin-binding growth factor with properties similar to those of conditioned medium from FGF-5-transfected eukaryotic cells.
  • the FGF-5 protein has 268 amino acids (corresponding to. the second open reading frame or "ORF 2" as disclosed in Goldfarb et al . , supra) the sequence of which is well-conserved across mammals.
  • the murine homologue shows 84% overall sequence identity to the human (Hebert et al . , Dev. Biol . 138:454-463 (1990)).
  • FGF-5 contained a hydrophobic N-terminal leader sequence typical of a secreted protein (Goldfarb et al . , supra) and was released into the medium of transformed NIH-3T3 cells (Bates et al . , Mol . Cell . Biol .
  • Ciliary neurotrophic factor promotes the survival of motor neurons in vitro and in vivo (PCT Publication No. WO 91/04316, 4/4/91, incorporated by reference herein) .
  • Gelfoam implants containing CNTF facilitated survival of motor neurons in severed facial nerves of newborn rats.
  • CNTF is a neurotrophic factor that exhibits biological activities that are very different from those exhibited by FGF-5, as described herein.
  • the present inventors have discovered that FGF-5 promotes the survival of motor neurons in vitro and is therefore useful for promoting motor neuron growth and survival. On this basis, it is expected that FGF-5 is useful in vivo for treating a disease associated with a deficiency in motor neuron number or function.
  • the present invention relates to a method of treating a motor neuron disorder comprising administering to a subject in need of such treatment an effective amount of FGF-5, or a functional derivative thereof that supports the survival of motor neurons.
  • the present invention is further directed to a method of treating a motor neuron disorder comprising administering to a subject in need of such treatment an effective amount of FGF-5, or a functional derivative thereof that supports the survival of motor neurons, in combination with an effective amount of at least one other agent that is capable of promoting motor neuron survival, growth, or differentiation.
  • the other agent is ciliary neurotrophic factor.
  • the other agent may be bFGF or a member of the brain-derived neurotrophic factor/ neurotrophin-3/ nerve growth factor (BDNF/NT-3/NGF) family of molecules, that supports the survival, growth and/or differentiation of motor neurons.
  • BDNF/NT-3/NGF brain-derived neurotrophic factor/ neurotrophin-3/ nerve growth factor
  • the above methods are useful for treating any motor neuron disorder such as those associated with trauma, amyotrophic lateral sclerosis, progressive spinal muscular atrophy, infantile muscular atrophy, juvenile muscular atrophy, poliomyelitis, post polio syndrome, or Charcot-Marie-Tooth disease.
  • the present invention is also directed to a pharmaceutical composition useful in the treatment of a motor neuron disorder comprising;
  • the pharmaceutical composition comprises, in addition to FGF-5 or a derivative thereof, at least one other agent that is capable of promoting motor neuron survival, growth, or differentiation.
  • a preferred other agent is ciliary neurotrophic factor.
  • a method of promoting motor neuron survival comprising exposing a motor neuron to an effective concentration of FGF-5 that is capable of promoting the survival of motor neurons.
  • the present invention is directed to a method of diagnosing a motor neuron disorder associated with an abnormal level of FGF-5 in a subject, comprising:
  • step (b) comparing the levels of FGF-5 in step (a) with the level of FGF-5 in an analogous sample from a normal individual or with a standard level of FGF-5. thereby detecting any abnormality in the level of the FGF-5 in the subject, the abnormality being diagnostic of the motor neuron disorder.
  • the disorder is preferably selected from among amyotrophic lateral sclerosis, progressive spinal muscular atrophy, infantile muscular atrophy, poliomyelitis, post-polio syndrome, Charcot-Marie Tooth disease, nerve trauma or nerve injury.
  • a method for assaying a test agent for its ability to alter the binding of FGF-5 to a motor neuron comprising:
  • the present invention is also directed to a method for assaying a test agent for its ability to enhance or inhibit the activity of FGF-5 in promoting the survival of motor neurons, comprising: (a) exposing motor neurons to a concentration of FGF- 5 effective in promoting the survival of the neurons;
  • the present invention further provides for animal models of motor neuron disease produced by immunizing an animal to FGF-5 or derivatives thereof or by administering to an animal anti-FGF-5 antibodies or antisense oligonucleotides specific for FGF-5.
  • Figure l shows Northern blots of RNA hybridized with a 500 bp cRNA probe to FGF-5.
  • Panel A 16 ⁇ g of total RNA from embryonic (E15 and E17) , newborn (NB) and adult (A) rat skeletal muscle. Blot exposed for 7 days.
  • Panel B Comparison of the size of the FGF-5 transcript in 5 ⁇ g total RNA obtained from E15 rat skeletal muscle (E15) and adult rat hippocampus (H) . Blot exposed .for 3 days. The positions of the FGF-5 specific message and the 18S and 28S ribosomal RNA bands are indicated by arrows.
  • Figure 2 is a graph showing the effects of increasing concentration of recombinant human FGF-5 on survival of cultured E6 chick motor neurons in the absence ( o ) and presence ( • ) of heparin (500 ng/ml) . Results are means ⁇ 1 standard deviation from a minimum of 4 cultures from at least 2 independent experiments.
  • Figure 3 is a graph showing a comparison of maximum motor neuron survival activity and synergism of FGF-5 with CNTF and other members of the FGF family.
  • Factors were added to cultures in concentrations previously determined to promote maximal survival: FGF-5, aFGF, hst/K-FGF - 100 ng/ml; bFGF - 20 ng/ml; CNTF - 1 ng/ml. Results are means from 6 determinations from three independent experiments. Error bars represent 1 standard error of the mean (SEM) . * Significantly different survival compared to cultures containing bFGF alone, p ⁇ 0.00005. ** Significantly different from cultures containing CNTF alone, p ⁇ 0.005 (Student's t-test) .
  • Figure 4 is a graph showing motor neuron survival activity of increasing concentrations of recombinant human FGF-5 prior to ( • ) or following immunoprecipitation with either anti-FGF-5 ( o ) or "control" anti-CNTF ( D ) serum.
  • Figure 5 is a graph showing motor neuron survival activity of E17 muscle extracts.
  • Column A Control, no muscle extract.
  • Columns B-D Addition of 250 ⁇ g/ml PBS-soluble extract prior to (column B) and following immunoprecipitation with anti-FGF-5 (column C) or anti-CNTF (column D) antisera.
  • Columns E-G Addition of 70 ⁇ g/ml high molar salt/EDTA extract prior to
  • Figure 6 is a graph showing motor neuron survival activity of adult muscle extracts.
  • Column A Control, no muscle extract.
  • Columns B-D Addition of 125 ⁇ g/ml PBS-soluble extract prior to (column B) and following immunoprecipitation with anti-FGF-5 (column C) or anti-CNTF (column D) sera.
  • Columns E-G Addition of 125 ⁇ g/ml PBS-soluble extract prior to (column B) and following immunoprecipitation with anti-FGF-5 (column C) or anti-CNTF (column D) sera.
  • Columns E-G
  • FGF-5 AS A TROPHIC FACTOR FOR SPINAL _ MOTOR NEURONS _
  • FGF- 5 is a major survival factor, present in skeletal muscle, for cultured spinal motor neurons and plays a role in the maintenance of motor neuron survival during development and in adulthood. Because of this activity, FGF-5 is useful in methods for promoting survival of motor neurons and in the treatment of diseases and disorders associated with loss of functional motor neurons.
  • FGF-5 as described in Goldfarb, M. et al . , PCT Publication WO 88/01767, Dec. 1, 1988, hereby incorporated by reference in its entirety) is used to promote the survival of motor neurons.
  • a naturally occurring mammalian FGF-5 is used.
  • a recombinant mammalian FGF-5 is used.
  • a chemically synthesized mammalian FGF-5 protein or glycoprotein is used. Methods for the synthesis of polypeptides of desired sequence on solid phase supports and their subsequent separation from the support are well-known in the art.
  • the preferred FGF-5 protein or glycoprotein useful in the methods and compositions of the present invention is of human origin.
  • the naturally occurring FGF-5 protein or glycoprotein used according to the present invention is preferably substantially free of other proteins or glycoproteins with which it is natively associated. "Substantially free of other proteins or glycoproteins” indicates that the FGF-5 protein or glycoprotein has been purified away from at least 90% (on a weight basis), and from even at least 99%, if desired, of other proteins and glycoproteins with which it is natively associated, and is therefore substantially free of them.
  • Such purification can be achieved by subjecting cells, tissue or fluid containing FGF-5 to a standard protein purification technique, for example, immunoaffinity chromatography using an immunoadsorbent column to which is immobilized a monoclonal antibody ( Ab) which binds to the protein.
  • FGF-5 glycoprotein is purified using a combination of standard methods, such as ammonium sulfate precipitation, molecular sieve chromatography, heparin affinity chromatography and ion exchange chromatography.
  • standard methods such as ammonium sulfate precipitation, molecular sieve chromatography, heparin affinity chromatography and ion exchange chromatography.
  • a mammalian FGF-5 protein or glycoprotein of the present invention can be biochemically purified from a variety of cell or tissue sources.
  • tissues such as embryonic skeletal muscle, especially of human origin, are preferred.
  • FGF-5 protein can be synthesized substantially free of other proteins or glycoproteins of mammalian origin in a prokaryotic organism or in a non-mammalian eukaryotic organism, if desired.
  • a recombinant FGF-5 protein or glycoprotein produced in mammalian cells such as transfected COS, NIH-3T3, or CHO cells, for example, is preferably a naturally occurring protein sequence. Where a naturally occurring FGF-5 protein or glycoprotein is produced by recombinant means, it is provided substantially free of the other proteins and glycoproteins with which it is natively associated.
  • a functional derivative of FGF-5 By “functional derivative” is meant a “fragment,” “variant,” “analogue,” or “chemical derivative” of the protein, which terms are defined below.
  • a functional derivative retains at least a portion of the function of the FGF-5 protein, for example reactivity with an antibody specific for the FGF-5 protein, the ability to bind to spinal motor neurons, the ability to promote growth or survival of spinal motor neurons which evidences its utility in accordance with the present invention.
  • fragment is used to indicate a polypeptide which is derived from FGF-5, preferably human FGF-5, and has naturally occurring protein sequence. Such a fragment may be produced by proteolytic cleavage of the full-length protein.
  • the fragment is obtained recombinantly by appropriately modifying the DNA sequence encoding the FGF-5 protein or glycoprotein to delete one or more amino acids at one or more sites of the C-terminal, N- terminal, and within the native sequence.
  • Fragments of a FGF-5 protein or glycoprotein are also useful for screening for compounds that are antagonists or agonists of FGF-5 action. It is understood that such fragments may retain one or more characterizing portions of the native protein or glycoprotein.
  • a preferred retained characteristic for use according to this invention is the capacity to promote spinal motor neuron growth or survival.
  • Another functional derivative intended within the scope of the present invention is a FGF-5 variant with amino acid deletions, insertions, substitutions or a combination thereof, relative to the naturally occurring protein.
  • Such variants may be derived from a naturally occurring FGF-5 protein or glycoprotein by appropriately modifying the DNA coding sequence to add codons for one or more amino acids at one or more sites of the C-terminal, N-terminal, and within the native sequence. It is understood that such a variant having additional amino acids retains one or more characterizing portions of the native FGF-5 protein or glycoprotein, as described above.
  • a preferred variant is one which has substituted amino acids, the variant being derived from a naturally occurring FGF-5 by appropriately modifying or mutating the DNA coding sequence to substitute one or more amino acids at one or more sites of the C-terminal, N-terminal, and within the native amino acid sequence. It is understood that such a variant having substituted amino acids retains one or more characterizing portions of the native FGF-5.
  • a preferred group of variants is that in which at least one amino acid residue in the protein molecule, and preferably, only one, has been removed and a different residue inserted in its place.
  • substitutions which may be made in the protein or peptide molecule of the present invention may be based on analysis of the frequencies of amino acid changes between a homologous protein of different species, such as those presented in Table 1- 2 of Schulz et al . (supra ) and Figure 3-9 of Creighton (supra ) . Base on such an analysis, conservative substitutions are defined herein as exchanges within one of the following five groups:
  • Substantial changes in functional or immunological properties are made by selecting substitutions that are less conservative, such as between, rather than within, the above five groups, which will differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or
  • substitutions are (a) substitution of Gly and/or Pro by another amino acid or deletion or insertion of Gly or Pro; (b) substitution of a hydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobic residue, e.g.. Leu, lie, Phe, Val or Ala; (c) substitution of a Cys residue for (or by) any other residue;
  • substitution of a residue having an electropositive side chain e.g., Lys, Arg or His, for (or by) a residue having an electronegative charge, e.g. , Glu or Asp; or (e) substitution of a residue having a bulky side chain, e.g., Phe, for (or by) a residue not having such a side chain, e.g., Gly.
  • deletions and insertions, and substitutions according to the present invention are those which do not produce radical changes in the characteristics of the FGF-5 protein or peptide molecule.
  • a variant typically is made by site-specific mutagenesis of the protein-encoding nucleic acid, expression of the variant nucleic acid in recombinant cell culture, and, optionally, purification from the cell culture, for example, by immunoaffinity chromatography using a specific antibody on a column (to absorb the variant by binding to at least one epitope) .
  • the activity of the cell lysate or a purified or semi-purified protein variant can be screened in a suitable screening assay for the desired characteristic. For example, a change in the immunological character of the protein peptide molecule, such as binding to a given antibody, is measured by a standard immunoassay. Biological activity is screened in an appropriate bioassay, as described herein. Modifications of such properties as redox or thermal stability, hydrophobicity, susceptibility to proteolytic degradation or the tendency to aggregate with carriers or into multimers are assayed by methods well known to the ordinarily skilled artisan. Any combination of deletion, insertion, and substitution may also be made to arrive at the final construct of a FGF-5 functional derivative, provided that the final construct possesses at least one desired activity or function of the intact FGF-5 protein or glycoprotein, as described above.
  • a functional derivative of FGF-5 glycoprotein with amino acid deletions, insertions or substitutions may be conveniently prepared by direct chemical synthesis, using methods well-known in the art.
  • a "chimeric" FGF-5 molecule constructed from FGF-5 in which one or more specific amino acid sequences are replaced with functionally active sequence(s) from another protein or peptide. Preferably the sequences are replaced with homologous sequences.
  • a "chemical derivative" of the FGF-5 protein or FGF-5 peptide- contains additional chemical moieties not normally a part of the protein.
  • Covalent modifications of the protein or peptide are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
  • Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines) , such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo-3(5-imidozoyl) propionic acid, chloroacetyl phosphate, N- alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2- chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2- oxa-1,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain.
  • Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues.
  • Other suitable reagents for derivatizing ⁇ -amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; 0- ethylisourea; 2,4 pentanedione; and transa inase- catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pK a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group.
  • tyrosyl residues are well-known in the art, in particular modifications which introduce spectral labels by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Carboxyl side groups are selectively modified by reaction carbodiimide (R'-N-C- N-R*) such as l-cyclohexyl-3-(2-morpholinyl(4-ethyl) carbodiimide or l-ethyl-3-(4-azonia-4,4- dimethylpentyl) carbodiimide.
  • carbodiimide R'-N-C- N-R*
  • aspartyl and glutamyl residue are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues.
  • these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Derivatization with bifunctional agents is useful for cross-linking the FGF-5 protein or peptide to a water-insoluble support matrix or to other macromolecular carriers.
  • cross-linking agents include, for example, l,l-bis(diazoacetyl)-2- phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate) , and bifunctional maleimides such as bis-N-maleimido-l,8-octane.
  • Derivatizing agents such as methyl-3-[p-azidophenyl) dithiolpropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • Such derivatized moieties may improve the solubility, absorption, biological half life, and the like.
  • the moieties may alternatively eliminate or attenuate any undesirable side effect of the protein and the like.
  • Moieties capable of mediating such effects are disclosed, for example, in Remington ' s Pharmaceutical Sciences , 16th ed. , Mack Publishing Co., Easton, PA (1980).
  • the biological activity of FGF-5 or of a functional derivatives thereof may be measured as motor neuron growth-promoting activity or differentiation-promoting activity, using any method known in the art.
  • increased survival of motor neurons may be measured by the method set forth in Arakawa et al. , J. Neurosci . 10:3507-3515 (1990)).
  • Increased sprouting of motor neurons may be detected by methods set forth in Pestronk et al . (1980, Exp. Neurol . 70:65-82) or Brown et al. (1981, Annu . Rev. Neurosci . 4:17-42).
  • an assay is performed as described in Section 6, below.
  • Increased production of motor neuron-associated molecules may be measured by bioassay, enzymatic assay, immunoassay, Northern blot assay, etc. , depending on the molecule to be measured.
  • Motor neuron dysfunction can be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, defective motor neuron conduction velocity, or functional disability.
  • FGF-5 may be combined with a second agent in order to support motor neuron survival and/or differentiation.
  • the second agent is also effective in promoting motor neuron survival and/or differentiation.
  • FGF-5 is combined with CNTF in order to promote motor neuron survival and/or differentiation.
  • FGF-5 combined with CNTF exerts at least an additive effect on motor neuron survival.
  • the present invention provides for methods of treatment of a motor neuron disorder comprising administering, to a subject in need of such treatment, an effective amount of FGF-5, or a functional derivative thereof, that supports the survival, growth or differentiation of motor neurons.
  • motor neuron disorder refers to any condition that disturbs the normal function of motor neurons. Such disorders may or may not specifically or even selectively affect motor neurons.
  • motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, toxin exposure, physical or surgical trauma, degenerative disease or malignant disease that affects motor neurons as well as any other components of the nervous system.
  • the methods of this invention may also be used to treat disorders that selectively affect motor neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome) , poliomyelitis, post polio syndrome and Hereditary Motorsensory Neuropathy (Charcot-Marie- Tooth Disease) .
  • motor neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio-Londe syndrome) , poliomyelitis, post polio syndrome and Hereditary Motorsensory Neuropathy (Charcot-Marie- Tooth Disease) .
  • Effective doses of FGF-5 for therapeutic uses discussed above may be determined using methods known to one skilled in the art. Effective doses, as well as supraoptimal or toxic doses, if they exist, may be determined, preferably in vitro , in order to identify the optimal dose range (see section 5.5, below).
  • FGF-5 may also be administered in combination with an effective amount of at least one other agent that is, itself, capable of promoting motor neuron survival, growth, or differentiation.
  • FGF-5 may be administered with CNTF or with a member of the BDNF/NT-3/NGF family, including but not limited to BDNF, NGF, NT-3, NT-4 and NT5.
  • FGF-5 and CNTF are co-administered in the treatment of a motor neuron disorder. Since soluble heparin potentiates the survival effect of FGF-5 on motor neurons, another embodiment provides administering FGF-5 in combination with heparin.
  • the preferred dose of heparin is one which achieves a tissue concentration of between about 10 ng/ml and 10 ⁇ g/ml, more preferably about 1 ⁇ g/ml.
  • FGF-5 a functional derivative of FGF-5, such as a peptide fragment thereof. Also intended is the use of agonists or antagonists of FGF-5.
  • the FGF-5 or other neurotrophic factor(s) of the invention may be administered in any pharmaceutically acceptable carrier.
  • the administration route may be any mode of administration known in the art, including but not limited to intravenously, intrathecally, subcutaneously, by injection into involved tissue, intraarterially, intranasally, orally, or via an implanted device.
  • an aqueous solution of FGF-5 is administered by subcutaneous injection.
  • Each dose may range from about 0.5 ⁇ g to about 50 ⁇ g FGF-5 per kilogram body weight, or more preferably, from about 3 ⁇ g to 30 ⁇ g FGF-5 per kilogram body weight.
  • the dosing schedule for subcutaneous administration of FGF-5 may vary from once a week to daily depending on a number of clinical factors, including the type of disease, severity of disease, and the subject's sensitivity to FGF-5.
  • Nonlimiting examples of dosing schedules are 3 ⁇ g/kg administered twice a week, three times a week or daily; a dose of 7 ⁇ g/kg twice a week, three times a week or daily; a dose of 10 ⁇ g/kg twice a week, three times a week or daily; or a dose of 30 ⁇ g/kg twice a week, three times a week or daily.
  • Effective doses of additional neurotrophic factors administered in combination with FGF-5, such as CNTF are in the same dose range as the effective doses of FGF-5 described herein.
  • One or more additional drugs may be administered in combination with FGF-5 to alleviate such undesired side effects, for example, an anti-pyretic, anti- inflammatory or analgesic agent.
  • the dose or frequency of FGF-5 administration may be decreased or increased to a "maintenance dose" which maintains the subject's clinical status at a desired level. If a subject develops resistance to FGF-5, the dose may be increased.
  • Clinical effects of FGF-5 therapy may be determined using methods well-known in the art for the particular disease, including tests of nerve function, muscle function, etc. Such tests may be used to monitor a subject's response to therapy and thereby optimize the dosing schedule during initial and maintenance phases of treatment. In the case of more severe disease, it may be preferable to administer doses such as those described above by alternate routes, including intravenously or intrathecally. Continuous infusion of FGF-5 may also be appropriate.
  • FGF-5 may result in either systemic or localized distribution of the FGF-5 (and any co-administered neurotrophic factor) depending on the route of administration and the dose.
  • intravenous or intrathecal administration of FGF-5 in place of, or in addition to, subcutaneous administration may be preferable.
  • an implant containing FGF-5 may be placed in or near the affected area. Suitable implants include, but are not limited to, gelfoam, wax, or microparticle-based implants.
  • FGF-5 may also be administered via a cellular, solid or semi-solid implant to achieve blood levels of FGF-5 similar to those attained by subcutaneous administration.
  • a cellular implant may comprise cells naturally producing, or genetically altered to produce, FGF-5, which secrete FGF-5 in vivo in a subject following inoculation.
  • the present invention also provides pharmaceutical compositions comprising an amount of FGF-5 effective to promote motor neuron survival and effective to .treat a disease associated with motor neuron dysfunction, in a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising an effective amount of FGF-5 together with one or more additional neurotrophic factors, preferably CNTF or a member of the member of the BDNF/NT-3/NGF family, in a pharmaceutically acceptable carrier.
  • the present invention provides for a method of promoting motor neuron survival, growth, and/or differentiation comprising exposing motor neurons to an effective concentration of a FGF-5.
  • This method roay b e carried out in vivo (see Section 5.4, above) or in vitro .
  • an effective amount of FGF-5, or a combination of FGF-5 with one or more neurotrophic factors can be determined on a case-by- case basis, as motor neurons from different tissue sources or from different species may exhibit different sensitivities. For any particular culture or cell type, it is desirable to obtain a dose- response curve that correlates FGF-5 concentration and motor neuron response.
  • motor neuron survival it may be useful to compare motor neurons exposed to FGF-5 to motor neurons not exposed to such factor, using, for example, vital dyes to evaluate survival, phase-contrast microscopy and/or neurofilament stain to measure neurite sprouting, or techniques that measure the bioactivity of motor neuron-associated compounds, such as the enzyme choline acetyJtransferase (CAT) .
  • vital dyes to evaluate survival
  • phase-contrast microscopy and/or neurofilament stain to measure neurite sprouting
  • neurofilament stain to measure neurite sprouting
  • bioactivity of motor neuron-associated compounds such as the enzyme choline acetyJtransferase (CAT) .
  • CAT choline acetyJtransferase
  • CAT activity is measured, for example, by harvesting and lysing treated or untreated motor neurons in a 20 mM Tris-HCl (pH 8.6) solution containing about 0.1% Triton X-100, removing an aliquot of several microliters, and measuring CAT activity.
  • Enzymatic activity is determined using, as a substrate, 0.2 ml [1- 14 C] acetyl-CoA, 300 mM NaCl, 8 mM choline bromide, 20 mM EDTA, and 0.1 mM neostig ine in 50 mM NaH 2 P0 4 (pH 7.4) buffer, using the micro-Fonnum procedure as described in Fonnum, 1975, J . Neurochem . 24:407-409, incorporated by reference in its entirety herein.
  • motor neurons may be prepared and cultured as described in the Examples, below, which exemplify chick and rat motor neurons. The skilled artisan will readily appreciate how to use these methods, with or without modifiction, for the culture of motor neurons of other species, including humans, without undue experimentation.
  • Chick spinal motor neurons are prepared by first dissecting lateral motor columns from the lumbar region of several E6 chick spinal cords, followed by trypsin treatment and trituration. The resultant cell suspension is filtered and centrifuged through a bed of 6.8% metrizamide. Cells from the interface are collected and the suspension concentrated by centrifugation, resuspended in C0 2 -buffered L-15 medium supplemented with horse serum (10%) and plated, preferably in 4-well culture dishes (CA. Greiner und S ⁇ hne GmbH, Niirtingen, FRG) at a concentration of about 1000-2000 cells/well. The culture wells are previously coated with poly-DL-ornithine and laminin, the latter solution being removed just prior to cell plating.
  • the cells are incubated at 37°C in a humidified 5% C0 2 incubator for 1 hour, test factors and/or extracts added at the appropriate concentration, and the cells are returned to the incubator for the length of the experiment.
  • the culture medium and factors may be augmented or replaced after 24 hours. Large phase-bright cells (>14 ⁇ m in diameter) are counted about 72 hours after plating (unless otherwise specified) in predetermined areas corresponding to 23% of each well bottom.
  • FGF-5 is added alone or with another one or more neurotrophic factors.
  • Preferred final concentrations of FGF-5 are between about 1 ng/ml and 300 ng/ml, more preferably about 100 ng/ml.
  • FGF-5, at the above concentrations may be added in conjunction with CNTF at a final concentration of at least about 1 ng/ml and preferably about 10 ng/ml.
  • the motor neuron cultures may then be maintained in serum-free defined medium at 37°C in a 95% air/5% C0 2 atmosphere at nearly 100% relative humidity.
  • the present invention also provides for a method of diagnosing a motor neuron disorder in a subject, comprising comparing the level of FGF-5 in a sample from the subject with an analogous sample from a normal individual, or with a standard level previously determined from normal individuals, and thereby detecting an aberrancy in the level of FGF-5 in the subject which positively correlates with the presence of the motor neuron disorder.
  • Motor neuron disorders that may be diagnosed in this manner include but are not limited to those listed above.
  • the neurotrophic factor level may be detected by any method known in the art including, but not limited to, immunoassays, preferably enzyme-linked immunoabsorbent assay (ELISA) , such as a "sandwich"- type ELISA, or any other assay that utilizes an antibody specific for FGF-5, including Western blot analysis and in situ hybridization. Also useful are measurements of biological activity of FGF-5 or molecular measurements such as Northern blot analysis, etc.
  • immunoassays preferably enzyme-linked immunoabsorbent assay (ELISA) , such as a "sandwich"- type ELISA, or any other assay that utilizes an antibody specific for FGF-5, including Western blot analysis and in situ hybridization.
  • ELISA enzyme-linked immunoabsorbent assay
  • any other assay that utilizes an antibody specific for FGF-5, including Western blot analysis and in situ hybridization.
  • measurements of biological activity of FGF-5 or molecular measurements such as Northern blot analysis, etc
  • Samples may be derived from any appropriate tissue or fluid of a subject, including but not limited to, nerve or brain tissue or cerebrospinal fluid or blood.
  • An aberrancy in FGF-5 level may be defined as a statistically significant difference in the level of FGF-5 in the subject compared to a normal control.
  • the aberrant level may be increased or decreased relative to normal levels, and/or may exist throughout the subject or only in a localized area.
  • Subjects exhibiting a decrease in FGF-5 may particularly benefit from administration of the factor, alone or in combination with another agent which promotes motor neuron growth, differentiation or survival.
  • Subjects exhibiting an increase in neurotrophic factor may be expressing a structurally abnormal FGF-5 or may suffer from an effective paucity of FGF-5 receptors.
  • Subjects with fewer than normal, or abnormal, FGF-5 receptors may be identified by testing cells of such subjects for their levels or activities of FGF-5 receptor, for example assessing the ability of such cells to bind detectably labeled FGF-5 and comparing the binding to that of cells from a normal, healthy individual.
  • Subjects with fewer or abnormal receptors may or may not benefit from supplemental FGF-5 therapy, and may particularly benefit from treatment with an FGF-5 agonist or analogue.
  • the present invention provides assay systems useful for identifying compounds that can treat motor neuron disorders. Such assay systems evaluate a test agent for the ability to block FGF-5 binding to motor neurons. Accordingly, the present invention provides a method for assaying a test agent for its ability to promote the survival, growth, or differentiation of motor neurons comprising exposing motor neurons in culture to detectably labeled FGF-5 capable of binding to the cells in the presence of the test agent. Competitive inhibition of FGF-5 binding by the test agent indicates that the test agent may be used in the treatment of motor neuron disorders.
  • a test agent identified in such an assay system is preferably further evaluated in vitro or in vivo for its ability to promote the survival, growth, or differentiation of motor neurons in culture.
  • FGF-5 may be detectably labeled by any method, including by the incorporation of radiolabelled amino acids, or by the addition of a detectable exogenous label.
  • a label may be fluorescent, may have enzymatic activity, may be an antibody that binds to FGF-5, or may be an antigen capable of binding to a detecting antibody.
  • radiolabelled FGF-5 together with an unlabeled test agent are incubated with motor neurons in culture under conditions that allow FGF-5 binding.
  • an identical motor neuron culture is exposed to radiolabelled FGF-5 in the absence of the test agent.
  • the cells are then washed to remove unbound FGF-5, and the radioactivity associated with the cells is measured. If the test agent binds to the FGF-5 receptor on the neurons, and competitively inhibits binding of labeled FGF-5, cells incubated with the test agent will have less radioactivity than the control cells.
  • the present invention further provides for animal model systems for motor neuron disorders comprising animals that have systemic or generalized depletion of FGF-5.
  • Generalized depletion of FGF-5 may be achieved by producing an animal that contains or produces antibody directed toward FGF-5.
  • animals are immunized with FGF-5 from another species or with FGF-5 that has been chemically modified to increase its immunogenicity.
  • FGF-5 may be achieved by introducing anti-FGF-5 antibody locally, e.g., by implanting a hybridoma cell line producing the antibody in a location where it is desirable to deplete FGF-5 activity.
  • Antibodies useful in these animal models are antibodies, polyclonal, monoclonal, chimeric, other others, which neutralize the biological activity of FGF-5, preferably, its motor neuron growth promoting or survival promoting activity.
  • FGF-5 IS A MAJOR MUSCLE- DERIVED SURVIVAL FACTOR FOR CULTURED SPINAL MOTOR NEURONS
  • FGF-5 a member of the FGF family, acts as a physiologically relevant neurotrophic factor and promotes motor neuron survival.
  • FGF-5 a member of the FGF family, acts as a physiologically relevant neurotrophic factor and promotes motor neuron survival.
  • FGF-5 mRNA is present in embryonic skeletal muscle during the period of naturally-occurring motor neuron death as well as in adult muscle. Extracts of these muscles had activity in promoting motor neuron survival.
  • An antibody to FGF-5 removed most of this activity from "the muscle extracts.
  • FGF- 5 is a target-derived trophic factor for spinal motor neurons.
  • Recombinant human FGF-5 was purified from the soluble fraction of E . coli lysates by heparin- Sepharose chromatography and Mono-S fluid phase liquid chromatography (FPLC) .
  • FPLC Mono-S fluid phase liquid chromatography
  • Recombinant rat CNTF was prepared from transfected E . coli using a modification of the method described by Masiakowski et al . , J. Neurochem . , 57:1003-1012 (1991) ,. hereby incorporated by reference) , in which the chromatographic purification of the protein on DEAE cellulose was replaced by a single reverse-phase HPLC step on a semi-preparative, butyl column (Baker) .
  • Recombinant human K-FGF was provided by Dr. Claudio Basilico, New York University, New York, USA.
  • Recombinant aFGF and bFGF were gifts from Dr. W. Risau, Martinsried, FRG.
  • N-fluorenylmethoxycarbonyl (Fmoc) amino acid derivatives 2-(lH-benzotriazol-l-yl)-1, 1, 3, 3- tetramethyluronium hexafluorophosphate (HBTU) , HMP resin, solvents and miscellaneous reagents for peptide synthesis were from Applied Biosystems, Rothstadt, FRG. Freund's complete and incomplete adjuvants, Leibovitz's L-15 medium were from GIBCO BRL, Eggenstein, FRG. Metrizamide was from Serva, Heidelberg, FRG. General laboratory reagents were from Sigma Chemie, Deisenhofen, FRG and E. Merck, Darmstadt, FRG.
  • Hindlimb skeletal muscle was dissected from embryonic (E15 and E17) , newborn and adult rats killed by ether overdose, taking particular care to exclude bone, cartilage and skin, and total RNA extracted from the muscle according to the method of Chomczynski et al . , Anal . Biochem . 162:156-159 (1987)). 20 ⁇ g of each RNA sample (determined spectrophotometrically) were glyoxylated and electrophoresed through a 1.4% agarose gel (Lindholm et al . , J. Biol . Chem .
  • the probe (specific activity 10 9 cpm/ ⁇ g) was made by run-on transcription of a pBluescript SK + vector containing a 2.2 kb mouse Fgf-5 cDNA fragment (Haub et al . , supra) linearized with Bglll.
  • the nonadecapeptide CFKQSEQPELSFTVTVPEK [SEQ ID NO:l] comprises a relatively hydrophilic 18 amino acid sequence from a C-terminal part of human FGF-5 (Phe 217 - Lys 234 ) . This sequence is highly conserved between animal species (100% identity between human and mouse) but bears no homology to other members of the FGF family.
  • the above peptide was synthesized on an Applied Biosystems 431A automated peptide synthesizer using N- Fmoc amino acid derivatives and HBTU assisted coupling steps. Following deprotection and cleavage from the resin, the crude peptide free acid was purified as required by reverse phase HPLC from 0.1% TFA on a butyl column using an acetonitrile gradient (0-70% acetonitrile in 30 min.).
  • New Zealand rabbits were immunized by intracutaneous injection into at least four sites on the back with 700 ⁇ g of the peptide dissolved in 500 ⁇ l water and emulsified with an equal volume of Freund's complete adjuvant. Rabbits were boosted with a similar quantity of peptide solution emulsified with Freund's incomplete adjuvant at three to four weekly intervals. Blood samples were obtained from an ear vein one week after boosting, allowed to coagulate (at 37°C for 2 hours then overnight at 4°C) , centrifuged (at 6000 x g, 25 min,)* Tne resultant serum was sterile filtered (0.2 ⁇ m filters) and used in immunoprecipitation assays.
  • Chick spinal motor neuron cultures were prepared as previously described (Arakawa et al . , supra) .
  • lateral motor columns were dissected from the lumbar region of six E6 chick spinal cords, trypsinized and triturated.
  • the resultant cell suspension was filtered and centrifuged through a bed of 6.8% metrizamide. Cells from the interface were collected and the suspension concentrated by centrifugation, resuspended in C0 2 -buffered L-15 medium supplemented with horse serum (10%) and plated in 4- well culture dishes (CA. Greiner und S ⁇ hne GmbH, Nurtingen, FRG) at a concentration of 1000-2000 cells/well.
  • the wells had been previously coated with poly-DL-ornithine and laminin, the latter solution being removed just prior to plating.
  • Hindlimb skeletal muscle was dissected from embryonic (E17) and adult rats as described above. Extracts of soluble skeletal muscle proteins were obtained ' by homogenizing the muscle on ice in two to three volumes of PBS in a glass-glass homogenizer and centrifugation (100,000 x g, 4°C, 30 min.). Matrix-bound components were obtained by re- extraction of the pellets obtained above with 2 or 3 initial muscle volumes of PBS supplemented with 3M NaCl and 10 mM EDTA, followed by centrifugation as above. The protein concentration of the resultant extracts was determined by the method of Bradford
  • the protein G Sepharose-antiserum complex was washed three times with 500 ⁇ l TBS, after which 20 ⁇ l 0.5 x TBS and either (a) 60 ⁇ l muscle extract (5 ⁇ g/ ⁇ l) or (b) 60 ⁇ l recombinant human FGF-5 solution (1 ng/ ⁇ l in L-15(C0 2 ) medium supplemented with 10% horse serum) was added. Following centrifugation, the resultant supernatants were tested for survival promoting activity by adding them to motor neuron cultures.
  • FGF-5 mRNA was expressed in the target tissue of motor neurons either during embryonic development or in adulthood.
  • Northern blot analysis was performed on total RNA obtained from hindlimb skeletal muscle of embryonic (E15 and E17) , newborn and adult rats.
  • a cRNA probe from mouse FGF-5 was found to specifically hybridize to a band migrating marginally faster than the 18S band in rat skeletal muscle RNA at all timepoints studied ( Figure 1) .
  • the probe also hybridized to a band of similar size in RNA prepared from other rat tissues, including adult hippocampus (D. Lindholm, supra) , a known site of FGF-5 expression in the mouse (Haub et al . , supra) .
  • FGF-5 The ability of recombinant human FGF-5 to support the survival of E6 chick motor neurons in vitro was determined in the absence and presence of heparin (Figure 2) .
  • FGF-5 supported the survival of a maximum of 27% of the initially-plated motor neurons at a concentration of 10 ng/ml after 72 hours in culture.
  • heparin sulphate 500 ng/ml
  • the survival effect of FGF-5 was increased significantly, such that 45% of plated motor neurons were supported at a concentration of 100 ng/ml.
  • Motor neurons cultured in the presence of the same concentration of heparin were not significantly different than control cultures.
  • FGF-5 The concentration of FGF-5 required to give half maximal survival (EC 50 ) of motor neurons was 800 pg/ml.
  • FGF-5 was significantly better than other heparin-dependent members of the FGF family which were tested, aFGF and hst/K-FGF (maximally 35% and 32% survival, respectively, 3 days; Figure 3) .
  • FGF-5 was less effective in promoting neuronal survival than were either bFGF or CNTF (maximally 50% and 61% survival, respectively, 3 days) .
  • FGF-5 did not support the survival of cultured E8 chick ciliary ganglion neurons.
  • the antiserum was raised in rabbits by immunization with a synthetic peptide representing a sequence from the C-terminal portion of human FGF-5.
  • the sequence was judged by hydropathy analysis (Hopp et al . , Proc . Natl . Acad . Sci . 78:3824-3828 (1981); Kyte et al . , J. Mol . Biol . 157:105-132 (1982)) to be relatively hydrophilic and therefore likely to be exposed on the surface of intact FGF-5.
  • the peptide spans a region of the C-terminal "tail" of FGF-5 which, although well-conserved between species, is not present in the other known members of the FGF family. This reduced the likelihood that the resultant antibodies would cross-react with other members of the FGF family.
  • Figure 5 shows the motor neuron survival activity of an extract of phosphate buffered saline- (PBS-) soluble components of embryonic (E17) rat skeletal muscle before (column B) and after (column C) the addition of FGF-5 antiserum which had been immobilized on protein G-Sepharose. Following centrifugation of the antiserum-Sepharose complex, the survival activity of the soluble embryonic muscle extract on E6 chick spinal motor neurons at the maximum protein concentration tested (250 ⁇ g/ml) was reduced from 60% to 36%. This represents a 47% reduction in survival activity, given the background survival of 9% in control cultures ( Figure 5, column A) .
  • PBS- phosphate buffered saline-
  • FGF-5 was responsible for a significant proportion of the survival activity of the PBS-soluble components of E17 rat skeletal muscle. Because, members of the FGF family, including FGF- 5, tend to bind to components of the extracellular matrix (ECM), we tested whether embryonic rat skeletal muscle contained additional, matrix-bound, FGF-5-like motor neuron survival activity which was not extractable with PBS alone. Thus, the pellets obtained after PBS extraction of E17 muscle were re- extracted with PBS containing 3M NaCl and lOmM ethylenedia ine tetraacetic acid (EDTA) . These conditions would be expected to disrupt interactions between ECM and molecules bound to it, allowing solubilization of matrix-bound components.
  • ECM extracellular matrix
  • the high molar salt extract of E17 muscle had a weaker survival promoting effect on cultured chick spinal motor neurons than did the PBS extract (approximately 37% survival; Figure 5, column E) . Notably, this effect was achieved at a lower total protein concentrations (60 ⁇ g/ml) .
  • PBS-soluble extracts of adult skeletal muscle exhibited maximal motor neuron survival activity of approximately 37% at a protein concentration of 125 ⁇ g/ml (3 day culture; Figure 6, column B) .
  • PBS-soluble extract of embryonic muscle none of the activity could be precipitated with anti- FGF-5 antiserum ( Figure 6, column C) .
  • FGF-5 A specific antiserum to FGF-5 was used to test whether the motor neuron survival activity of the muscle extracts was attributable to FGF-5.
  • hindlimb skeletal muscle of E17 rats an age at which naturally-occurring embryonic motor neuron death is underway; Oppenhei , R.W. , J. Comp . Neurol . 246 : 281- 286 (1986)
  • approximately 60% of the total maximal extractable survival activity was immunoprecipitated by the anti-FGF-5 antiserum.
  • This immunoprecipitable activity was distributed between a PBS-soluble form and a form which required high molar salt and EDTA for extraction.
  • FGF-5 activity in adult skeletal muscle was exclusively in the matrix-bound form.
  • FGF-5 like the other members of the FGF family, exhibits strong binding to the glycosaminoglycan heparin (Zhan et al . , supra) and thus would be expected to be tightly bound to heparin sulphate-like components of the ECM.
  • the greater resistance to solubilization of the FGF-5 activity from adult muscle compared to embryonic muscle reflects a difference in the nature or composition of the ECM in the adult animal.
  • FGF-5 activity in E17 skeletal muscle extracts could originate from two sources: the muscle itself and/or a site of earlier expression.
  • the observed expression of FGF-5 mRNA in this tissue is support for the presence of FGF-5 in muscle. Indeed, in the mouse, the most prominent embryonic expression of FGF- 5 mRNA at times beyond gastrulation occurs at E12.5 to E14.5 in a patch of mesenchyme at the base of the hindlimb (Haub et al . , supra) .
  • FGF-5 protein expressed in the developing rat may therefore be deposited in the surrounding muscle, thus contributing to the observed FGF-5-like activity in the E17 muscle extract.
  • FGF-5 mRNA in adult rat muscle is a strong indication that the muscle itself is the major source of FGF-5 in this tissue.
  • The- previously reported absence of FGF-5 mRNA in Northern blots of RNA from adult mouse skeletal muscle suggests either a difference in regulation of the FGF-5 gene in these two species, or that the amount of FGF-5 mRNA in mouse fell below their detection limits.
  • the latter interpretation is supported by the fact that levels of FGF-5 mRNA in adult rat skeletal muscle are lower than in some central nervous system sites, such as the hippocampus (D. Lindholm, supra) .
  • a family of membrane-spanning receptor tyrosine kinases are capable of acting as receptors and signal-transducing units for FGFs (Ruta et al . , Oncogene 3:9-15 (1988); Kornbluth et al . , Mol . Cell . Biol . 8:5541-5544 (1988); Lee et al . , Science 245:57-60 (1989); Dionne et al . , EMBO J . 9:2685-2692 (1990); Pasquale, E.B., Proc . Natl . Acad . Sci . USA 87:5812-5816 (1990); Miki et al.
  • FGF-5 Recombinant human FGF-5 is able to bind and activate FGF receptors 1 and 2 (D. Clements, et al . , Oncogene , in press, (1993)).
  • FGF-5 and bFGF towards these receptors correlate with the relative concentrations of these factors required to support chick motor neurons in culture, suggesting that the expression of FGF receptor 1 or 2 in embryonic motor neurons partially accounts for FGF survival effects.
  • FGF-5 chick motor neurons cultured in the presence of both FGF-5 and bFGF
  • at least three distinct FGF receptors may participate in motor neuron survival, one receptor with specificity for FGF-5, one for bFGF and a third (perhaps FGF receptor 1 or 2) able to respond to both factors.
  • the molecular nature of the motor neuron survival promoting activity present in embryonic and adult skeletal muscle extracts that could not be immunoprecipitated by anti-FGF-5 antiserum remains undetermined.
  • the presence in skeletal muscle of other FGF-related molecules, though not well characterized at the molecular level, is known.
  • bFGF-like immunoreactivity has been detected in developing skeletal muscle of embryonic chick (Joseph-Silverstein et al . , J . Cell Biol . 108:2459-2466 (1989)), rat (Gonzalez et al . , J. Cell Biol . 110:753-765 (1990)) and rabbit (Morrow et al . , J . Clin . Invest . 85:1816- 1820 (1990)), while a molecule with bFGF-like characteristics has been detected in human embryonic and adult skeletal muscle (Vaca et al. , J . Neurosci . Res . 23:55-63 (1989)). aFGF has been detected
  • aFGF and bFGF renders unlikely a role for either as a target-derived neurotrophic factor for motor neurons.
  • a number of other candidate target-derived trophic factors for developing spinal motor neurons have previously been proposed. Choline acetyltransferase development factor (CDF) , purified from rat skeletal muscle, increased levels of choline acetyltransferase enzyme activity in embryonic rat spinal cord cultures (McManaman et al . , J . Biol . Chem . 263:5890-5897 (1988); J . Neurochem . 53:1763-1771 (1989)).
  • CDF Choline acetyltransferase development factor
  • CDF Although conclusive survival promoting effects of CDF on highly-enriched or purified motor neuron cultures have not been reported, CDF shares some properties with FGF-5, including (1) an inability to support the survival of chick ciliary neurons in culture, (2) apparent abundance in skeletal muscle and (3) additive effects with bFGF (McManaman et a . , supra) . Nonetheless, several differences between CDF and FGF-5, such as extent of glycosylation and heparin-binding characteristics, suggest that they are distinct entities. Identification of the exact molecular nature of CDF awaits the cloning of this factor.
  • LIF leukemia inhibitory factor
  • FGF-5 indeed plays a role in regulating motor neuron survival in vivo , or perhaps partakes in the modulation of other motor neuron-related functions, such as transmitter synthesis or synapse formation.
  • Embryos were obtained from pregnant Wistar rats at day 15 of gestation following ether anesthesia and cervical dislocation of the mother. Ventral parts of the lateral spinal columns of 6 embryos were carefully subdissected and cut into small pieces using forceps. The spinal columns were then treated with 0.05% trypsin in HBSS(-) for 15 minutes, washed in HBSS(-) and gently triturated 5 or 6 times in 0.1% Soybean trypsin inhibitor with a 1 ml Gilson pipette. The cell suspension thus obtained was filtered through a 50 ⁇ m nylon mesh filter, and the trituration procedure was repeated with any remaining spinal cord fragments.
  • Detached cells were collected by aspiration, centrifuged and resuspended in culture medium prior to plating at a density of about 2000- 3000 cells per well of a 4 well tissue culture dish coated with poly-ornithine and laminin (Arakawa et al . , supra) . After incubating for 1 hour at 37°C in a 5% C0 2 atmosphere, neurotrophic and growth factors were added. Culture medium and factors were renewed after 24 hours in culture and thereafter every 48 hours. The total number of cells originally plated per well was determined after 4 hours. Neuron survival was assessed by counting surviving neurons in culture after 5 days. Results are presented as surviving cells as a % of originally plated cells.

Abstract

Procédés de traitement des affections des neurones moteurs, par exemple la sclérose latérale amyotrophique, consistant à administrer à un sujet ayant besoin d'un tel traitement une dose efficace d'un facteur neurotrophique appelé facteur de croissance des fibroblastes 5 (FGF-5), qui maintient la survie, la croissance et/ou la diférenciation des neurones moteurs. Une combinaison du FGF-5 et d'un second agent, de préférence le CNTF, est également utilisable dans le traitement des affections des neurones moteurs. On a également prévu des procédés permettant de favoriser la survie, la croissance et/ou la différenciation in vitro des neurones moteurs, des procédés de diagnostic, et des systèmes de dosage pouvant servir à identifier des agents ayant une activité semblable à celle du FGF-5, et utilisables dans le traitement des affections des neurones moteurs. En outre, on a prévu des compositions pharmaceutiques comportant le FGF-5 seul ou en combinaison avec un second agent maintenant la survie, la croissance et/ou la différenciation des neurones moteurs, ainsi qu'un excipient pharmaceutiquement acceptable.
PCT/EP1994/000764 1993-03-12 1994-03-11 Traitement des affections des neurones moteurs a l'aide du facteur de croissance des fibroblastes 5 (fgf-5) WO1994020125A1 (fr)

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WO1995015176A1 (fr) * 1993-12-01 1995-06-08 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procedes activant la duree de vie et facilitant la differentiation des sous-classes de neurones cholinergiques et serotonergiques a l'aide du facteur 5 de croissance des fibroblastes
WO1999045952A2 (fr) * 1998-03-12 1999-09-16 Genentech, Inc. Methode de prevention de la mort des neurones de la retine et de traitement d'affections oculaires
WO2001000662A2 (fr) * 1999-06-29 2001-01-04 Eli Lilly And Company Utilisation de polypeptides fgh-8 humains comme agents neurotrophiques
US6331523B1 (en) 1998-03-12 2001-12-18 Genentech, Inc. Method of enhancing the survival of retinal neurons and treating ocular diseases using FGF-5
EP1225909A2 (fr) * 1999-10-02 2002-07-31 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Facteur de croissance des fibroblastes 5 (fgf-5) dans un antigene de leucocyte t associe aux tumeurs
WO2003063893A3 (fr) * 2002-01-31 2004-04-08 Max Planck Gesellschaft Agonistes du fgfr
EP1788094A1 (fr) * 2004-08-24 2007-05-23 Eisai R&D Management Co., Ltd. Procede consistant à distinguer le type de neurone de la moelle epiniere en ciblant le gene de la proteine corli1

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995015176A1 (fr) * 1993-12-01 1995-06-08 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Procedes activant la duree de vie et facilitant la differentiation des sous-classes de neurones cholinergiques et serotonergiques a l'aide du facteur 5 de croissance des fibroblastes
AU754380B2 (en) * 1998-03-12 2002-11-14 Genentech Inc. Method of preventing the death of retinal neurons and treating ocular diseases
WO1999045952A2 (fr) * 1998-03-12 1999-09-16 Genentech, Inc. Methode de prevention de la mort des neurones de la retine et de traitement d'affections oculaires
WO1999045952A3 (fr) * 1998-03-12 2000-11-23 Genentech Inc Methode de prevention de la mort des neurones de la retine et de traitement d'affections oculaires
US6331523B1 (en) 1998-03-12 2001-12-18 Genentech, Inc. Method of enhancing the survival of retinal neurons and treating ocular diseases using FGF-5
WO2001000662A2 (fr) * 1999-06-29 2001-01-04 Eli Lilly And Company Utilisation de polypeptides fgh-8 humains comme agents neurotrophiques
WO2001000662A3 (fr) * 1999-06-29 2001-05-17 Lilly Co Eli Utilisation de polypeptides fgh-8 humains comme agents neurotrophiques
EP1225909A2 (fr) * 1999-10-02 2002-07-31 THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES Facteur de croissance des fibroblastes 5 (fgf-5) dans un antigene de leucocyte t associe aux tumeurs
WO2003063893A3 (fr) * 2002-01-31 2004-04-08 Max Planck Gesellschaft Agonistes du fgfr
US7531304B2 (en) 2002-01-31 2009-05-12 Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften E.V. Method for screening FGFR-4 agonists
CN100531796C (zh) * 2002-01-31 2009-08-26 马普科技促进协会 Fgfr激动剂
EP1788094A1 (fr) * 2004-08-24 2007-05-23 Eisai R&D Management Co., Ltd. Procede consistant à distinguer le type de neurone de la moelle epiniere en ciblant le gene de la proteine corli1
EP1788094A4 (fr) * 2004-08-24 2009-05-13 Eisai R&D Man Co Ltd Procede consistant à distinguer le type de neurone de la moelle epiniere en ciblant le gene de la proteine corli1

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IL108950A0 (en) 1994-06-24
ZA941729B (en) 1994-10-13

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